The major hypothesis of this proposal is that bacteria capable of sustained vaginal colonization of a broad cross-section of women can be engineered to produce alpaca- derived beta integrin-specific variable region antibodies (VHH) that will inhibit infection with HIV-1. This approach to preventing HIV-1 transmission offers the unique advantage of targeting receptor-ligand interactions that are completely independent of mutable viral proteins. A similar delivery system targeting Glycoprotein D of herpes simplex type 2 will also prevent infection with that virus, which enhances transmission of HIV-1 infection. This hypothesis will be pursued by demonstration of the efficacy of bacterially-delivered VHH using three different mouse model systems and two different bacterial strains, one of which, Streptococcus gordonii, is selected specifically for the purpose of demonstrating proof of principle in mouse model systems that require pre-treatment of mice with progesterone. Lactobacillus rhamnosus GR-1 will be used in a mouse model that does not require progesterone pre-treatment, as this strain of lactobacillus has been demonstrated to be effective in colonizing a broad cross-section of women. Lactobacilli are appealing as a microbicide vehicle because they are "generally regarded as safe" organisms by the FDA, constitute the primary bacterial component of the normal flora of the female genitourinary tract, intrinsically inhibit the growth of more virulent bacteria, would be transparent to users, and would allow dissociation of microbicide application from coitus. Alpaca-derived antibody binding domains (VHH) are developed from a class of camelid antibodies consisting solely of heavy chains and the binding determinants of which are single protein domains that are much smaller than any which might be generated from classical antibodies. Using this technology we will: 1) Raise antibodies in alpacas against the ectodomain of CD18, the beta-chain component of the CD11a/CD18 heterodimer that is LFA-1. From circulating PBMC of the immunized alpacas we will generate a library of VHH screened by phage display for binding affinity. Those VHH will then be expressed from plasmids and/or the bacterial chromosome of Lactobacillus rhamnosus GR-1 and Streptococcus gordonii. 2) Using the methods described in Aim 1 generate VHH targeting HSV-2 glycoprotein D. 3) Evaluate the potential toxicity of anti-CD18 and anti-GpD by measuring transepithelial resistance in two different in vitro model systems. 4) Test the ability of the CD18 and GpD-specific VHH to block transmission or neutralize infection in transwell or in vitro neutralization assays. 5) Evaluate colonization of the mouse vagina and secretion of CD18-specific VHH by transformed S. gordonii in progesterone-treated Hu-PBL-SCID mice and by L. rhamnosus in non-progesterone-treated humanized bone marrow liver thymus NOD- SCID (BLT) mice. Evaluate the ability of colonized mice to resist infection with cell- associated and cell-free HIV-1. 6) Evaluate colonization of the mouse vagina and production of Glycoprotein D-specific VHH by transformed S. gordonii progesterone- treated BALB/c mice. Evaluate in these mice the appearance of anti-Glycoprotein D and anti-CD18 VHH antibodies in vaginal lavage fluid and serum and examine vaginal lavage fluid for the appearance of inflammatory cells induced by the VHH. The passive protective ability of in situ-produced HSV-2 specific VHH will also be assayed in this mouse vaginal challenge model. and 7) If dictated by results of the previous studies, develop a plasmid-based expression system that does not depend on antibiotic selection. At the completion of these studies, we will have proved the potential efficacy of this approach using in vivo model systems and will have developed the VHH constructs that could be used in the clinical setting as well as established their production from a lactobacillus species that also may be applicable to the clinical setting.